Process for producing l-glutamic acid



United 3,096,252 PROCESS FOR PRODUCING L-GLUTAMIC ACKD Shinichi Motozaki, Tokyo, Toshinao Tsunoda and Shinii Okumura, Kanagawa-ken, Ryuichiro Tsugawa, Tokyo,

Patented July 2, 1963 scribed above for L-glutamic acid fermentation. In accordance with this invention, L-glutamic acid is produced by culturing L-glutamic acid producing bacteria belonging to the genus Brevibacterium under aerobic conditions Toshinofl Matsui Kumamotmken, and Atsuo Kitai and 5 in a culture medium containing at least one member se- Noboru Miyachi, Tokyo, Japan, assignors to Ajinolee-ted from the group consisting of desthiobiotin (5- moto Kabushiki Kaisha and Sanraku Shuzo Kabushiiri methyl-2-oxo-4-imidazolidine caproic acid) having the Kaisha, both of Tokyo, Japan, and both corporations fo l of Japan N0 Drawing. Apr. 19, Ser. No. HOOO(CHZ)4CHR OH NH Claims priority, application Japan Apr. 23, 1960 13 Claims. (Cl. 195-47) I O This invention relates to processes for producing L-glu- CH3 OH NH tamic acid, and more particularly to novel processes for biotimmsulfoxide having the forml 11a producing the same by fermentation techniques using 15 bacteria. CH2CHNH Various methods are known for producing L-glutamic so 0 acid by fermentation techniques employing L-glutamic- 4 CH 1 acid producing bacteria. For example, methods are known for producing L-glutamicacid 'by culturing bacih OOH teria under aerobic conditions in a culture medium containing carbohydrates such as glucose, fructose, sucrose, and blocytln having the formula maltose, starch hydrolysates or the like and nitrogen 0 sources such as inorganic or organic ammonium salts, g urea, ammonia solution, ammonia gas or the like as the primary components, and also a small amount of amino EN NH acids, vitamin B, and inorganic salts such as potassium l, H NH 2 phosphates, magnesium sulphate, iron and manganese A) ions and the like. In these conventional methods, howz (O 2)4CONH.QOI-Ig) CHOOOH ever, the rate of growth of the fermentation bacteria is s poor and the fermentation efliciency, i.e. the amount of L-gluta-mic acid produced per hour, is very low. Therealong with carbohydrates, nitrogen sources and inorganic fore, those conventional methods are uneconomic-a1 and salts as the primary components. unsuitable for producing L-glutamic-acid on alarge scale. In processes of this invention, the rate of growth of This is due to the poor growth of fermentation bacteria fermentation bacteria is accelerated, and the amount of 4 caused by a lack of nutrients required for their growth. L-glutarnic acid produced and accumulated is remarkably It is an object of this invention to provide an improved increased. process for producing L-glutamic acid wherein the amount As noted, although there exist lots of prior arts perof L-glutamic acid produced and accumulated in a cultaining to L-glutamic-acid-fermentation using bacteria, no ture medium is unexpectedly increased. It is another ob prior art reporting that the efficiency and yield of L-gluject of this invention to provide a process for producing tamic acid fermentation are outstandingly increased by L-glutamic acid eificiently and on an industrial scale. culturing said bacteria in a culture medium containing Other objects and advantages of this invention will be apat least one member from the group including desthiobioparent from the following detailed description thereof. tin, biotin-d-sulfoxide and biocytin is known.

It has now been discovered that L-glutamic acid can In case of fermentation using various strains belonging be efficiently produced with a high yield in a short time to the genus Brevi'bacterium capable of producing L-gluby culturing L-glutamic acid producing bacteria under. tamic acid,,the influence of the addition of desthiobiotin, aerobic conditions in the presence of at least one member biotin-d-sulfoxide and biocytin on the rate of fermentaselected from the group consisting of-desthio'b-iotin, biotintion and the amount of L-glutamic acid produced are d-sulfoxide, and biocytin in a culture medium such as degiven in the following tables.

TABLE I Bacterium used Brevibacterium lactofer- Brevibacterium saccharoly- Brevibacterium fltwum, Brem'bacterium immario- Brevibacterium roseum, mentum, ATOO No.13869 ticum, ATCO No. 14066 ATCC No. 14067 plrilium, A'IOO No. 14068 T00 No. 13825 dl-Dcsthio- Analysis biotin (7/ Amount Amount Amount Amount Amou t Residof Residof L- Besid- 0f Residof Residof Growth ual glntamic Growth ual glutarnic Growth ual glutamic Growth ual glutamic Growth ual glutamic sugar, acid prosugar, acid prosugar, acid prosugar, acid pr0- sugar, acid propercent duced, percent duccd, percent duced, percent duced, percent duced, percent percent percent percent percent 0.11 85.3 6.3 92.5 0.7 0.14 83.2 4.3 0.13 78.2 2.9 0.15 80.8 3.9 0. 59 31.9 30.4 0.46 36.4 21.2 0.70 17.2 37.6 0.65 10.2 36.0 0.52 25.8 39.5 0.52 33.2 17.0 0.50 34.4 32.0 0. 60;. 7.1 49.2 0. 60 4.5 29.1 0.73 1.9 40.8 .3% gag 0. 63 26.0 44.7 0.65 0.6 44.7 0.65 1.5 27.4 0.78 0.8 39.3

All of experiments described in Table I excluding experiments using Brevibacterz'um laczofermentum ATCC No. 13869 were carried out at a temperature of 30 C. for 40 hours under shaken culture in the culture medium consis-ting of the following ingredients:

, 4 t is apparent from the above tables that the fermenta tion of any strain belonging to the genus Brevibacterium proceeds inefficiently, that the rate of growth of the termentation bacteria is poor, and that a large amount of 5 residual sugar is left in the medium and therefore the Glu o "percent" 1 4 yield of L-glutamic acid is as low as in the absence KH PO d 1 of .desthiobiotin, biotin-d-sulfoxide or biocytin. MgSO -7H O d0 0.04 On the contrary, it is seen that in case of the presence Fe++ p.p.m 2 of at least one of said compounds, active fermentation, Mn++ p.p.rn 2 10 i.e., an increased rate of growth of fermentation bacteria, Hydroly'sate of soybean protein (total nitrogen and substantial consumption of the saccharide is effected,

2.4 g./.dl.) ml./d1 0.1 the amount of L-glutamic acid produced, i.e., the concen- Vltamm 1 hydrochloride --'Y 200 tration thereof, substantially exceeds 4.0 g./ dl. and a yield Urea "P of L-glutamic acid as high as about can be obtained. In case of Brevibacterium lactofermemum ATCC N Thus, the addition of desthiobiotin, biotin-d-sulfoxide or 13869, 50 7/1. of vitamin B hydrochloride and 09-32% biocytin has an obviously remarkable effect. of urea were used. In case desthiobiotin, biocytin or bioti-n-d-sulfoxide TABLE II Bacterium used Brevz'bacterium Zactofermentum, Brevibacterium saccharolyticum, Brevibacterium flat/um, Brevibucterium roseum,

ATCC No. 13869 ATCC No. 14066 ATCC No. 14067 ATCC N 0. 13825 Biotin-dsultoxide Analysis Amount of Amount of Amount of Amount of Residual L-glutamic Residual L-glutamic Residual L-glutamic Residual L-glutamic Growth sugar, acid, pro- Growth sugar, acid pro- Growth sugar, acid pro- Growth sugar, acid propercent duced, percent duced, percent duced, percent duced,

percent percent percent percent Bacterium used Brevz'bacterium lactofermentum,

Brezibacterium saccharolyticum,

Brevibacterium flavum, Brem'bacterium roseum,

ATCC No. 13869 ATCC No. 14066 ATCC No. 14067 ATCC No. 13825 Biocytin Analysis Amount of Amount of Amount of Amount of Residual L-glutamic Residual L-glutamic Residual L-glutamic Residual L-glutamic Growth sugar, acid, pro- Growth sugar, acid pro- Growth sugar, acid pro- Growth sugar, acid propercent duced, percent duced, percent duced, percent duced,

percent percent percent percent All of the experiments described in Table II were carried out at a temperature of 31 C. for 40 hours, the cul ture medium being shaken and consisting of the follow- In the above tables, Growth represents degree of turbidityin the culture medium diluted 26 times at 562 my, and percentages of Residual sugar and Amount of L-glutamic acid produced are the basis of the weight of raw saccharides charged.

is used alone, the amount is preferably not more than 20 7/1., not more than 15 v/l. or not more than 15 7/1. cor responding to said compounds respectively. Additionally, it is possible to achieve the same outstanding effect as described above by a combination of two or more of the group including desthiobiotin, biotin-d-sulfoxide and biocytin. With the combination of biotin and biocyt-in, the total amount thereof is preferably not more than 15 7/1. With the combination of desthiobiotin and biocytin or biotin-d-sulfoxide, the total amount thereof is preferably not more than 18 7/1.

The L-glutamic-acid producing bacteria belonging to the genus Brevibacter-ium which may be used in the present invention include Brevibacterium lactofermentum ATCC No. 13869, Brevibacterium saccharolyticum ATCC No. 14066, Brevibacterium flavum ATCC No. 14067, Brevibacterium immariophilium ATCC No. 14068 and'Brevibacterium roseum ATCC NO. 13825.

In the fermentation in accordance with the invention,

the culture is carried out while maintaining the pH of the culture medium at slightly alkaline conditions by an addition of ammonia or urea. Temperatures and time of culturing will depend upon the bacteria used.

The methods of fermentation which may be used in this invention include fermentation involving shaking or employing submerged culture under aeration. L-glutamic acid produced in the culture medium may be recovered with any conventional techniques, e.g., by filtering the fermented medium to remove the cells, concentrating the filtrate, adjusting the pH to 3.2 by the addition of hydrochloric acid and precipitating L-glutamic acid.

To illustrate the present invention, the following examples are given.

' Example 1 A culture medium containing glucose 10.39%, KH PO 0.1%, MgSO .7H O 0.04%, Fe++ 2 p.p.m., Mn++ 2 p.p.m., hydrolysate of soybean protein (total nitrogen 2.4 g./dl.) 0.1 ml./dl., vitamin B hydrochloride 50 'y/l. and dl-desthiobiot-in 7'y/l. was adjusted to pH of 7.0 and sterilized by steam heating. Then there was added 2% of sterilized urea solution (45 g./dl.) to the culture medium, and the cells of Brevibaetei'ium lactofermentum ATCC No. 13869 cultured in non-saccharide bouillon agar medium at 30 C. for 24 hours were inoculated followed by a shaking of the culture at 31 C., the shaking being effected at the rate of 120 pm. and 7 cm. amplitude. During the culturing, the decomposition of urea by urease results in ammonia and the pH was changed to alkalinity. However, the ammonia thus formed was gradually assimilated and consumed, and consequently the pH reaches 7.0 16 hours from the beginning of the culturing. At the end of this time, 2% of urea solution (45 g./dl.) was added and the fermentation was continued for 6 hours further. The pH was again decreased to 7.0 and 3% of urea solution (45 g./dl.) was added. The culturing was completed 40 hours from the beginning thereof. L-glutamic acid in the culture medium thus obtained amounted to 5.23 g./dl. and the yield was 51.3% on the basis of the weight of the raw saccharide charged.

Example 2 A culture medium containing glucose 10.2%, KH PO Mn++5 p.p.m., glycine 80 mg./l., dl-alanine 160 mg./l., L-methionine 8'0 mg./l., dl-phcnylalanine 160 mg./l., L-histidine 80 mg./l., vitamin B hydrochloride 100 'y/l., and dl-desthiobiotin 10 /1. was adjusted to a pH of 6.5 and sterilized at high pressure. There was added 4% of sterilized aqueous urea (45 g./dl.) to the medium and the cells of Brevibacterium saccharolyticum ATCC No. 14066 cultured in non-saccharide bouillon agar medium at 30 C. for 18 hours were inoculated followed by shaking at 2930 C. at the rate 125 rpm. and 7 cm. of amplitude. The culturing was completed 40 hours from the beginning thereof.

L-glutamic acid in the culture medium thus obtained amounted to 4.68 g./dl. and the yield was 45.8% on the basis of the weight of the raw saccharide charged.

Example 3 The culture medium containing glucose 10.4%, KH PO' 0.1%, MgSO .7H O 0.04%, Fe++ 2 p.p.m., Mn++ 2 p.p.m., hydrolysate of soybean protein (total nitrogen 2.4 g./dl.) 0.1 ml./dl., vitamin B hydrochloride 200 /1., and dl-desthiobiotin 10 'y/l. was sterilized at high pressure. There was added 2% of sterilized aqueous urea (45 g./dl.) to the medium and then the cells of Brevibacterium flavum ATCC No. 14067 cultured in non-saccharide bouillon agar at 30 C. for 24 hours were inoculated followed by shaking at 30 C. 2% and 3% of aqueous urea were added at 12 hours and 22 hours from the beginning of the culturing respectively. The culturing was completed 40 hours from the beginning thereof.

6 L-glutamic acid in the culture medium thus obtained amounted to 5.12 g./dl. and the yield was 49.3% on the basis of the weight of the raw saccharide charged.

Example 4 Example 3 was repeated except that Brevibacterium roseum ATCC No. 13825 was used, and 2% and 3% of aqueous urea were added to the medium 14 hours and 22 hours from the beginning of the culturing.

L-glutamic acid in the fermented medium thus obtained amounted to 5.18 g./dl. and the yield was 49.8% on the basis of the weight of the raw saccharide charged.

Example 5 Example 3 was repeated except that Brevibacterium immariophilium ATCC No. 14068 was used, and 2% and 3% of aqueous urea were added to the medium 14 hours and 21 hours from the beginning of the culturing.

L-glutamic acid in the fermented medium thus obtained amounted to 3.03 g./dl. and the yield was 29.1% on the basis of the raw saccharide charged.

Example 6 A medium which had a pH of 7.0 and contained glucose 1 0%, KH PO 0.1%, MgSO .7H O 0.04%, Fe++ 2 p.p.m., Mn++ 2 p.p.m., hydrolysate of soybean protein (total nitrogen 2.4 g./dl.) 0.1 ml./dl., vitamin B hydrochloride 200 'y/l., and biotin-d-sulfoxide 8 'y/l. was sterilized by steam. There was added 2% of aqueous urea (45 g./dl.) to the medium and the cells of Brevibacterium lactofermentum ATCC No. 13869 cultured in bouillon agar for 24 hours were inoculated followed by shaking at 3 1 C. for 40 hours. Dining the culturing, the pH was raised to about 9.0 and then decreased to 7.0. 2% of aqueous urea and then 3% of aqueous urea were added.

L-glutamic acid in the fermented medium thus formed was 4.63 g./dl. and the yield was 46.3% on the basis of the raw sacchmide charged.

Example 7 Example 6 was repeated except that Brevibacterium saccharolyzicum ATCC No. 14066 was used and the concentration of biotin-d-sulfoxide was 12 v/l.

L-glutamic acid in the fermented medium thus obtained amounted to 4.21 g./dl., and the yield was 42.1% on the basis of the weight of the raw saccharide charged.

Example 8 Example 6 was repeated except that Brevibacterium flavum ATCC No. 14067 was used and the concentration of biotin-d-sulfoxide was 6 'y/l.

L-glutamic acid in the fermented medium thus obtained amounted to 5.19 g./l., and the yield was 51.9% on the basis of the weight of the raw saccharide charged.

Example 9 Example 6 was repeated except that Brevibacterium roseum ATCC No. 13825 was used, and the concentration of biotin d-sulfoxide was 5 1 /1.

L-glutamic acid in the fermented medium thus obtained amounted to 4.61 g./dl., and the yield was 46.1% on the basis of the weight of the raw saccharide charged.

Example 10 Example 6 was repeated except that Brevibacterium immarz'ophilium ATCC No. 14068 was used, the concentration of biotin-d-sulfoxide was 6 'y/l., and 2% and 3% of aqueous urea were added 13 hours and 18 hours from the beginning of the culturing.

L-glutamic acid in the fermented medium thus obtained amounted to 2.43 g./dl., and the yield was 24.3% on the basis of the weight of the raw saccharide charged.

Example 11 Example 6 was repeated except that 6 /1. of biocytin was used in lieu of 8 7/1. of biotin-d-sulfoxide.

L-glutamic acid in the fermented medium thus obtained was 4.23 .g./dl., and the yield was 42.3% based on the weight of the raw saccharide charged.

Example 12 Example 7 was repeated except that 16 'y/ 1. of biocytin was used in lieu of biotin-d-sulfoxide.

L-glutamic acid in the fermented medium thus obtained was 3.6 g./dl., and the yield was 36% based on the weight of the raw saccharide charged.

Example 13 Example 8 was repeated except that 8 'y /l. of biocytin was used in lieu of biotin-d-sulfoxide.

L-glutarnic acid in the fermented medium thus obtained amounted to 4.86 g./dl., and the yield was 48.6% based on the weight of the saccharide charged.

Example 14 Example 9 was repeated except that 6 v/l. of biocytin was used in lieu of biotin-d-sulfoxide.

L-glutamic acid in the fermented medium thus obtained amounted to 4.85 g./d1., and the yield was 48.5% based on the weight of the raw saccharide charged.

Example 15 Example 10 was repeated except that 6 'y/ 1. of biocytin was used in lieu of biotin-d-sulfoxide, and 2% and 3% of aqueous urea were added 14 hours and 23 hours later respectively.

L-glutamic acid in the fermented medium thus obtained amounted to 2.99 g./dl., and the yield was 29.9% based on the weight of the raw saccharide charged.

We claim:

1. In a process for producing L-glutamic acid which comprises culturing L-glutamic acid producing bacteria belonging to the genus Brevibacterium under aerobic conditions in a culture medium containing carbohydrates, nitrogen sources, amino acids, and inorganic salts toproduce and accumulate L-glutamic acid in said medium, the improvement comprising culturing the said bacteria under aerobic conditions in said medium in the presence of at least one member selected from the group consisting of desthiobiotin, biot-in-d-sulfoxide and biocytin, and recovering L-glutamic acid thus formed.

2. Process according to claim 1 wherein the said L- glutamic-aoid producing bacterium is Brevibacterium lactofermentum.

3. Process according to claim 1 wherein the said L- glutamic-acid producing bacterium is Brevibacterium saccharolyticum.

4. Process according to claim 1 wherein the said L- glutamic-acid producing bacterium is Brevibacterium flavum.

5. Process according to claim 1 wherein the said L- glutamic-acid producing bacterium is Brevibacterium rosewm.

6. Process according to claim 1 wherein the said L- glutarn-ic-acid producing bacterium is Brevibacterium immariophilium.

7. Process according to claim 1 wherein the said culture medium contains 320 'y/-l. of desthiobiotin.

8. Process according to claim 1 wherein the said culture medium contains 2-15 'y/l. of tbiotin-d-sulfoxide.

9. Process according to claim 1 wherein the said culture medium contains 2-15 'y/l. of biocytin.

10. Process according to claim 1 wherein the culture medium includes a total amount of desthiobiotin and biotin-d-sulfoxide of 3-18 1 /1.

11. Process according to claim 1 wherein the culture medium includes a total amount of desthiobiotin and biocytin of 3l8 'y/l.

12. Process according to claim 1 wherein the culture medium includes a total amount of biotin-d-sulfoxide and b-iocytin of 215 'y/l.

13. Process according to claim 1 wherein the culture medium includes a total amount of desthiobiotin, biotind-sulfoxide and biocytin of 1-18 'y/l. 

1. IN A PROCESS FOR PRODUCING L-GLUTAMIC ACID WHICH COMPRISES CULTURING L-GLUTAMIC ACID PRODUCING BACTERIA BELONGING TO THE GENUS BREVIBACTERIUM UNDER AEOBIC CONDITIONS IN A CULTURE MEDIUM CONTAINING CARBOHYDRATES, NITROGEN SOURCES, AMINO ACIDS, AND INORGANIC SALTS TO PRODUCE AND ACCUMULATE L-GLUTAMIC ACID IN SAID MEDIUM, THE IMPROVEMENT COMPRISING CULTURING THE SAID BACTERIA UNDER AEROBIC CONDITIONS IN SAID MEDIUM IN THE PRESENCE OF AT LEAST ONE MEMBER SELECTED FFROM THE GROUP CONSISTING OF DESTHIOBIOTIN, BIOTIN-D-SULFOXIDE AND BIOCYTIN, AND RECOVERNG L-GLUTAMIC ACID THUS FORMED. 